Specific neuronal transduction by recombinant scAAV2-GFP.

<p>Following injections of scAAV2-GFP into the sensorimotor cortex (A–D), red nucleus (RN) (E–H) and DRG (I–L), strong expression of GFP (green) was detected. The sections from these three areas were stained for NeuN (red) to specifically label the neurons (C, G and K). Scale Bars: 500 µm (A–E and I) and 200 µm (F–H, J–L).</p

Implication of scAAV2-GFP for labeling CST axons after injury.

<p>Dorsal column lesion resulted in a corresponding loss of CST axons, as visualized by anterograde transport of scAAV2-GFP at 4 weeks after viral injection into the sensorimotor cortex (A and C). GFAP staining of the section was to label the lesion site and cavity (B and E). Low magnification images shown in A–B, Corresponding high-magnification images shown in D–F. Scale Bars: 500 µm (A–C), 200 µm (D–F).</p

Comparison of transgene expression of scAAV2 with ssAAV2 vectors in DRG.

<p>A mixture of scAAV2-GFP and ssAAV2-mCherry vectors was injected into the cervical level DRGs of adult rats. DRG were analyzed one week after viral delivery. GFP (A and D) and mcherry (B and E) expressed simultaneously in prepared DRG samples. Some neurons in DRG and dorsal root axons were only clearly labeled with the scAAV2-GFP vector, and not with the ssAAV2-mCherry vector (arrows in C and F). Scale Bars: 200 µm (A–C), 100 µm (D–F).</p

GFP expression in DRG and labeling of central and peripheral axons.

<p>GFP expressed in DRG neurons of the cervical spinal cord after scAAV2-GFP intraganglion injections (A–C). βIII-tubulin staining showed labeling in both neurons and axons (B and C). Many GFP positive fibers are visible in the dorsal horn (D and E), the dorsal roots (F), and can be traced over long distances to the cervical level of spinal cord (G and H). The peripheral nerve was also brightly labeled (I). Scale Bars: 200 µm (A–C, F and I), 500 µm (D–E, G–H).</p

Implications of scAAV2-GFP labeling in dorsal root axons after crush.

<p>GFP-labeled axons were examined after dorsal root crush injury. In un-injured animals, GFP-labeled axons enter the spinal cord from the entry zone along with other axons (A and B). After dorsal root crush, cross sections of injured dorsal root with attached spinal cord showing GFP-expressing axons stopped in the DREZ (G and H). DREZ boundary is approximated with a dashed line. Scale Bars: 500 µm (A and C), 200 µm (B and D).</p

GFP expression in RN and anterograde labeling of RST.

<p>GFP expression was observed in the RN following scAAV2-GFP injection, as illustrated in coronal sections (A and B). The schematic inset in panel A illustrates the coordinates and levels of this section. Higher magnification images demonstrate anterograde transport of GFP to the contralateral site (C), midbrain (D and E) and cervical spinal cord in coronal (F and G) and sagittal sections (H and I). The box frames in panels D and F represent the regions magnified in panels E and G, respectively. Scale Bars: 500 µm (AH), 200 µm (I).</p

Schematic overview of experimental procedures.

<p>Schematic representations show pathways for the CST, RST and central axons of the DRG. A: The CST originates from the sensorimotor cortex, decussates at the medullary level of the brain stem and descends in the deep layer of the dorsal column. scAAV2-GFP vector was injected into sensorimotor cortex (green stars indicate GFP expression). Dorsal column lesion (red line in A) was performed to transect fibers of the CST in the cervical spinal cord. B: The RST runs from the neuronal bodies in the brainstem, crosses to the contralateral side and descends in the lateral white matter of the spinal cord. scAAV2-GFP was injected into the red nucleus (RN) to label the RST. C: Central axons of DRG originate from DRG neurons, pass through the dorsal root entry zone and ascend in the spinal cord dorsal column to reach the brain stem. Red lines in C indicate dorsal root crush and dorsal column lesions that transect the central axons of DRG in the dorsal root and spinal cord respectively. scAAV2-GFP was injected into DRG to anterogradely label central axons of the DRG.</p

GFP expression in the sensorimotor cortex and anterograde labeling of CST axons.

<p>A shows the bright field of low-power image of sensorimotor cortex and corresponding low-power and high-power fluorescence images demonstrate high transduction efficiency of GFP (B and C). The schematic inset in panel A illustrates the coordinates and levels of this section<b>.</b> GFP positive CST fibers were found 4 weeks after viral injections above the corpus callosum (D and E), in the medulla (F and G), in the cervical spinal cord, in coronal (H) and sagittal (I) sections. The frame in the panel F shows a low magnification of the pyramidal tract, box represent high magnification in panel G. Animals used for panels A–E and H–I received bilateral viral injection. Animal for panels F and G received unilateral viral injections. Scale Bars: 500 µm (A, B and H), 200 µm (C, D and F) and 100 µm (E, G and I).</p

Combining scAAV2-GFP with BDA tracer for labeling sensory and motor axons after injury.

<p>Following injections of scAAV2-GFP into the DRG and BDA into the sensorimotor cortex, sections of cervical spinal cord were examined in normal (A–D) and dorsal column lesion animals (E–L). GFAP staining labeling the lesion cavity are shown in blue (G and K). scAAV2-GFP-labeled dorsal sensory axons are shown in green (right of the lesion) (E and I) and BDA labeled CST fibers are shown in red (left of the lesion) (F and J). Scale Bars: 500 µm (A–H), 200 µm (I–L).</p

Probing the Carboxyester Side Chain in Controlled Deactivation (−)‑Δ⁸‑Tetrahydrocannabinols

We recently reported on a controlled deactivation/detoxification approach for obtaining cannabinoids with improved druggability. Our design incorporates a metabolically labile ester group at strategic positions within the THC structure. We have now synthesized a series of (−)-Δ⁸-THC analogues encompassing a carboxyester group within the 3-alkyl chain in an effort to explore this novel cannabinergic chemotype for CB receptor binding affinity, in vitro and in vivo potency and efficacy, as well as controlled deactivation by plasma esterases. We have also probed the chain’s polar characteristics with regard to fast onset and short duration of action. Our lead molecule, namely 2-[(6a<i>R</i>,10a<i>R</i>)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6,9-trimethyl-6<i>H</i>-dibenzo­[<i>b</i>,<i>d</i>]­pyran-3-yl]-2-methyl-propanoic acid 3-cyano-propyl ester (AM7438), showed picomolar affinity for CB receptors and is deactivated by plasma esterases while the respective acid metabolite is inactive. In further in vitro and in vivo experiments, the compound was found to be a remarkably potent and efficacious CB1 receptor agonist with relatively fast onset/offset of action